Proper regulation of the cell cycle transition from the resting phase G1, to the DNA synthetic phase, S, is often lost in cancer cells. This can result from the expression of viral oncoproteins, such as the E6 and E7 proteins encoded by the high risk human papillomaviruses (HPVs), which abrogate the functions of the tumor suppressor proteins p53 and pRB and, thus, mechanistically contribute to human carcinogenesis. Indeed, the importance of this function of E6 and E7 is underscored by the observation that cervical cancer cells that do not express these viral oncoproteins have suffered inactivating mutations in p53 and pRB. It is now clear that several different genetic events can lead to inactivation of these tumor suppressors in different tumor types. For example, cyclin D1 or cdk4 over expression is an alternative to pRB inactivation in some cancers, and deletion of p16/INK/4A, a negative suppressor of cyclin D/cdk4 function, is also a common alternative to pRB loss. This work has defined a pathway of alternative biochemical targets in cancer cells, alteration of any one of which may suffice to significantly dysregulate the G1-to-S phase transition. Our preliminary results suggest that cdk6 is also a potential oncogene acting in the pRB pathway, since it is preferentially activated in oral cancers. Furthermore, we have shown that over expression of cdk6 can render cells insensitive to p53-mediated G1 arrest, in part by interfering with the function of the cdk inhibitor p21 cip1/WAF1. The work described in this proposal is designed to identify and test the role of novel human papilloma viruses unique to oral squamous carcinomas and to identify alterations in the G1 cell cycle control machinery that contribute to tumor progression. In particular, the oncogenic role of cdk 6 will be investigated in oral cancer cell lines. These goals will be achieved by 1) identifying the biological and biochemical activities of HPVs associated with oral carcinomas; 2) determining the status of the p53 and pRB pathways in normal and neoplastic cultures of oral epithelial cells; and 3) testing the role of cdk6 in oral epithelial neoplasia through the construction of viral vectors encoding activated or dominant- negative cdk6.